PROJECT SUMMARY Gut microbiota play critical roles in determining health, and the structure and function of these microbiota are dependent on contemporary and historical host-microbe interactions. This application proposes research to understand processes relating to the normal homeostasis between hosts and gut microbes. Gut microbiota establishment and development is dependent on many factors, including innate and adaptive immunity, inter- microbe interactions, and diet. Microbiota composition and stability are crucial to health, and disruption has serve disease and pathogen infection consequences. A balance must be struck between rapid innate immune responses to limit pathogen invasion, while moderating responses against beneficial coevolved mutualists. Questions remain about how host immunity can, (1) maintain long-term associations with host-adapted core microbes, but (2) contribute to microbiota perturbation during development, leading to dysbiosis and health- related functional changes. The bumblebee gut microbiota model in this proposal provides an excellent opportunity to gain a broad understanding of microbiota structure and function determined by host-microbe interactions via innate immunity. The system is: (i) relatively simple, (ii) well described, (iii) unlike other insect models, e.g. Drosophila, reflective of some key human-gut microbiota interactions, and (iv) accessible to experimental manipulation, with available genomic and transcriptomic tools for hosts and microbes. The overarching proposal goal is to test a hypothesis of immune-mediation of the gut microbiota, and elucidate how host innate immunity can both disrupt microbiota structure, with functional consequences, and, together with reciprocal coevolved interactions with microbes, determine long-term dynamics of specific host-microbe associations. The first aim studies how host immune status (determined by experimental inoculation mimicking pathogenic infection) affects gut microbiota structure and beneficial functioning, to be determined by MiSeq sequencing and an infection protocol to assay the gut microbiota's protective function. This aim also assesses the persistence of immune-mediated perturbation and the ability of social interactions to resurrect dysbiotic microbiomes. The second main aim focuses on long-term specific associations between hosts and core microbes, and the role of innate immunity in facilitating these interactions. Two non-exclusive hypotheses will be tested: (a) host immune recognition and responses allow the persistence of beneficial core microbes, and (b) long-term associations are facilitated by microbe resistance to host immune effectors. Comparisons will be made between core native host bacteria and those from divergent host lineages in the induction of immune responses on experimental establishment and the ability to resist host immune components. Research on host-microbiota interactions is highly integrative, conceptually and technically. Thus, the proposed research fulfills a primary AREA program goal by exposing undergraduate students to technique-rich research based on a hypothesis driven framework, which will enhance understanding of a topic of clear biomedical importance.